Phosphatidic acid is the primary second messenger generated from the receptor-mediated activation oi phospholipase D (PL-D). Various agents exert their activity through the activation of this pathway. We have recently shown that epidermal growth factor (EGF) stimulates PL-D activity in normal rat osteoblastic cells increasing phosphatidic acid production. In addition, we have previously demonstrated that EGF increase cytosolic Ca+2 in normal rat osteoblastic cells (Loza et al. 1995). The biochemical mechanisms by which these events occur are not completely understood. The purpose of our studies with Dr. Rosemary Dziak is tc determine the effects of phosphatidic acid on cytosolic Ca+2 levels in osteoblastic cells, and to study the possible involvement of phosphatidic acid in the EGF dependent increase in cytosolic Ca+2 in these cells Primary cultures were obtained from fetal rat calvariae by sequential collagenase digestion, and grown in BGJ media until confluency. Cells were sub-cultured on round coversplips which were transferred to the stage of an inverted fluorescence microscope. Cytosolic Ca+2 measurements were performed in Fura-2A loaded cells using a SPEX Fluorog II spectrofluorometer. EGF (50 nM) significantly increased intracellular Ca+2 levels in a rapid and sustained manner. When the cells were stimulated with phosphatidic acid (2 mM) a transient and marked response was elicited. When cells were incubated in Ca+2 free media, the response elicited by phosphatidic acid was blocked. Similar blockage was observed when cells were pretreated with Verapamil (5 IlM). Following the stimulation with phosphatidic acid, the EGF response to increases in cytosolic Ca+2 appeared to be reduced to approximately 50%. These results stronglv suggest that phosphatidic acid participates in the regulation of cvtosolic calcium in osteoblastic cells. and that EGF's dependent activation oi phospholipase D is also involved in the regulation of this cation.